Excessive accumulation of triglycerides (triacylglycerols) in adipose tissue is associated with obesity, and in non-adipose tissues, such as skeletal muscle and liver, with insulin resistance. The final step in triglyceride synthesis, the covalent joining of diacylglycerol and long-chain fatty acyl CoA, is catalyzed by acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes. Two DGAT enzymes, the products of genes from different families, have been identified. The focus of this grant has been to elucidate the functions of these DGAT enzymes in cells, tissues, and physiology of whole animals. During the first funding period, we identified and cloned the DGAT1 and DGAT2 genes, and we discovered that mice lacking DGAT1 are resistant to diet-induced obesity and diabetes. In this renewal, we focus on two important outstanding questions concerning DGAT enzymes and triglyceride synthesis. Specific Aim 1 addresses: How does DGAT1 deficiency in mice increase energy expenditure and insulin sensitivity? We propose experiments to determine the mechanisms by which DGAT1 deficiency in WAT results in increased energy expenditure and increased insulin sensitivity in Dgat1-/- mice. These experiments will address the hypothesis that DGAT1 deficiency in WAT results in altered secretion of hormonal factors that regulate energy and glucose metabolism in other tissues. The last two aims address: Why are there two mammalian DGA T enzymes and what are their functional differences? Specific Aim 2 will determine the in vivo functions of DGAT2 by studying obesity susceptibility in Dgat2v/- mice and by generating tissue specific knockouts of DGAT2. Specific Aim 3 will determine the functional differences between DGAT1 and DGAT2 in cells, tissues, and whole animal physiology. In this aim, we will examine the intracellular Iocalizations of the two DGAT enzymes, and we will determine whether DGAT1 functions as an acyl CoA:retinol acyltransferase in vivo, a possibility raised by recently obtained preliminary data. These investigations should substantially enhance our understanding of DGAT enzymes and triglyceride metabolism, and they should further define the role of triglyceride synthesis in diseases such as obesity and diabetes.